Methane Production In Anaerobic Digestion Research Articles

Impact of Polyethylene-Glycol-Induced Water Potential on Methane Yield and Microbial Consortium Dynamics in the Anaerobic Degradation of Glucose.

This study investigated the relationship between water potential (Ψ) and the cation-induced inhibition of methane production in anaerobic digesters. The Ψ around methanogens was manipulated using polyethylene glycol (PEG) in a batch anaerobic reactor, ranging from -0.92 to -5.10 MPa. The ultimate methane potential (Bu) decreased significantly from 0.293 to 0.002 Nm3 kg-1-VSadded as Ψ decreased. When Ψ lowered from -0.92 MPa to -1.48 MPa, the community distribution of acetoclastic Methanosarcina decreased from 59.62% to 40.44%, while those of hydrogenotrophic Methanoculleus and Methanobacterium increased from 17.70% and 1.30% to 36.30% and 18.07%, respectively. These results mirrored changes observed in methanogenic communities affected by cation inhibition with KCl. Our findings strongly indicate that the inhibitory effect of cations on methane production may stem more from the water stress induced by cations than from their direct toxic effects. This study highlights the importance of considering Ψ dynamics in understanding cation-mediated inhibition in anaerobic digesters, providing insights into optimizing microbial processes for enhanced methane production from organic substrates.

Metagenome reveals the possible mechanism that microbial strains promote methanogenesis during anaerobic digestion of food waste

For better understanding the mechanism of microbial strains promoting methane production, four strains Hungatella xylanolytica A5, Bacillus licheniformis B1, Paraclostridium benzoelyticum C2 and Advenella faeciporci E1 were inoculated into anaerobic digestion systems. After bioaugmentation, the cumulative methane production of A5, B1, C2 and E1 groups elevated by 11.68%, 8.20%, 18.21% and 15.67% compared to CK group, respectively. The metagenomic analysis revealed that the species diversity and uniformity of the experimental groups was improved, and hydrolytic acidifying bacteria, represented by Clostridiaceae, Anaerolineaceae and Oscillospiraceae, and methanogens, such as Methanotrichaceae and Methanobacteriaceae, were enriched. Meanwhile, the abundance of key genes in carbohydrate, pyruvate and methane metabolism was increased in the inoculated groups, providing reasonable reasons for more methane production. The strengthening mechanism of microbial strains in this study offered a theoretical foundation for selecting a suitable bioaugmentation strategy to solve the problems of slow start-up and low methane production in anaerobic digestion.

Effect of bioaugmentation on gas production and microbial community during anaerobic digestion in a low-temperature fixed-bed reactor.

Low temperature is one of the limiting factors for anaerobic digestion in cold regions. To improve the efficiency of anaerobic digestion for methane production in stationary reactors under low-temperature conditions, and to improve the structure of the microbial community for anaerobic digestion at low temperatures. We investigated the effects of different concentrations of exogenous Methanomicrobium (10, 20, 30%) and different volumes of carbon fiber carriers (0, 10, 20%) on gas production and microbial communities to improve the performance of low-temperature anaerobic digestion systems. The results show that the addition of 30% exogenous microorganisms and a 10% volume of carbon fiber carrier led to the highest daily (128.15 mL/g VS) and cumulative (576.62 mL/g VS) methane production. This treatment effectively reduced the concentrations of COD and organic acid, in addition to stabilizing the pH of the system. High-throughput sequencing analysis revealed that the dominant bacteria under these conditions were Acidobacteria and Firmicutes and the dominant archaea were Candidatus_Udaeobacter and Methanobacterium. While the abundance of microorganisms that metabolize organic acids was reduced, the functional abundance of hydrogenophilic methanogenic microorganisms was increased. Therefore, the synergistic effect of Methanomicrobium bioaugmentation with carbon fiber carriers can significantly improve the performance and efficiency of low-temperature anaerobic fermentation systems.

Combined hydrothermal pretreatment of agricultural and forestry wastes to enhance anaerobic digestion for methane production

The dense structure and significant differences in the properties of agricultural and forestry wastes make it difficult to achieve anaerobic conversion. This study employed hydrothermal pretreatment (HTP) and combined hydrothermal pretreatment (CHTP) to enhance the anaerobic digestion (AD) performance of agricultural and forestry organic wastes. At a specific hydrothermal condition of 140℃ for 30 mins, the cumulative methane production of garden waste (GW), chicken manure (CM), duck manure (DM), and pig manure (PM) increased by 14.70%, 14.18%, 27.48%, and 21.81%, respectively. Under the same conditions, CHTP of garden waste with livestock and poultry manures showed noticeable synergistic effects, resulting in the methane production potential of garden waste mixed with chicken manure, duck manure, and pig manure increased by 28.82%, 36.59%, and 37.56%, respectively, compared with the untreated group. Metabolomic analysis showed that CHTP could significantly promote the decomposition of biomass carbohydrates, proteins, and lipids, leading to increased levels of small molecular metabolites. Analysis of microbial community diversity showed that HTP and CHTP increase the abundance of specific functional microbial communities, such as hydrolysis bacteria and methanogens, thereby promoting the anaerobic production of methane from biomass waste. Energy balance analysis shows that HTP-AD and CHTP-AD have a positive net energy benefit. This study provides an efficient CHTP technology and theoretical support for the resource utilization and energy conversion of agricultural and forestry wastes.

Optimum Magnetite–Zeolite Nanoparticles Loading on the Cathode of Microbial Electrochemical–Anaerobic Digestion System for Enhancing Methane Generation

Microbial electrochemical systems (MESs) can enhance methane production in anaerobic digestion, but their performance is hindered by inadequate electron transfer between the cathode and microbes. Magnetite–zeolite (MZ)‐based catalysts accelerate electron exchange; however, the optimal loading of MZ is crucial for maximizing MES performance. In this study, different loadings of the MZ catalyst ranging from 0 to 2 mg cm−2 on the cathode were evaluated to obtain an optimum loading for maximizing methane recovery from MES. Electrochemical analyses demonstrated enhanced electrochemical reduction kinetics of the cathode with increased MZ loading from 0 to 2 mg cm−2. Among the tested loadings, the MES with 1 mg cm−2 MZ on the cathode exhibited the highest methane production rate at 548 mL L−1 d−1, surpassing other MES operations with 2 and 0.5 mg cm−2 (499 and 434 mL L−1 d−1, respectively). Additionally, the MES with 1 mg cm−2 MZ demonstrated the highest soluble chemical oxygen demand removal efficiency of 87% and total coulomb recovery of 1444.06C ± 42.60C, with the lowest amount of volatile fatty acids accumulation. Consequently, MZ is recommended as a superior catalyst for enhancing MES performance and suggested to utilize 1 mg cm−2 MZ loading on the cathode to maximize methane production.

Zero-valent iron enhanced methane production of anaerobic digestion by reinforcing microbial electron bifurcation coupled with direct inter-species electron transfer

Zero-valent iron (ZVI) can facilitate methanogens of anaerobic digestion (AD). However, the impact of ZVI on the micro-energetic strategies of AD microorganisms remains uncertain. This study aimed to elucidate the development of an energy conservation model involving direct interspecies electron transfer (DIET) and electron bifurcate (EB) by using four types of ZVI. Overall, the ZVI addition resulted in a substantial increase in methane production (1.26 to 2.18 times higher), and the effect of boron (B) doped ZVI was particularly pronounced. The underlying mechanism may be the formation of energy harvest pathway related to DIET. In detail, B-doped ZVI could enhance its interfacial binding to cytochrome c. Decreased polar solvation energy from 20.473 to 1.509 kJ/mol is beneficial for electron transfer, thereby augmenting the flavin-bounded Cytc activity and DIET process. Besides, ZVI-enhanced EB enzyme activity like HdrA2B2C2-MvhAGD could improve the EB process, which can couple with DIET for electron transfer and energy conservation. Energy analysis based on EB-coupled DIET metabolism pathways demonstrated that the ATP saved in this coupled model theoretically line in 0.25 to 0.5 mol ATP/mol substrate. Overall, this study offers valuable insights into microbial energetic strategies pertaining to the utilization of conductive materials, with the target of enhancing methane recovery efficiency from organic waste.

Acyl homoserine lactone-based regulation strategy for improved methane production in anaerobic digestion of agricultural wastes

The acyl homoserine lactone (AHL)-based regulation strategy presents a considerable potential in improving anaerobic digestion (AD) efficiency of complex substrates. However, the reinforcement mechanisms are still unclear. In this study, the effects of different AHL types (C4-HSL, C7-HSL, C12-HSL) and different adding times (0 d, 5 d, 15 d) on AD performance of agricultural wastes (corn straw and cattle manure) were investigated. The results indicated that all types of AHLs exerted positive effects on AD, and the adding time rather than the AHL type was the key factor affecting AD processes. The C12-5d group obtained the highest accumulated methane production (310.7 ± 2.56 mL/g VS), which increased by 47.71% than that of the Control group. The exogenous AHLs facilitated the hydrolysis and acidification of complex substrates and the utilization of released low-molecular-weight organic acids in AD systems. Furthermore, the role of methanogenic archaea was upregulated and the dominance of bacteria was relatively weakened in AD systems, which favored the establishment of their balanced and symbiotic metabolic relationship. The enriched Methanobacterium strengthened the hydrogenotrophic methanogenesis pathway. This study provides new insights into the regulation strategy of AD for agricultural waste treatment based on AHL addition.

Effects of inocula on methane production and the microbial community in a rice straw anaerobic digestion system

Field experiments have shown that the mixed anaerobic digestion of straw with livestock and poultry manure can improve the efficiency of biogas production. We hypothesize that inoculation with livestock and poultry manure increase the efficiency of methane production by improving the diversity and structure of microbial community in the field anaerobic digestion system. In this study, an experimental system was established to simulate the microbial community of the mixed anaerobic digestion system in the field. The methane production and taxonomic composition of bacterial and archaeal communities in the rice straw anaerobic digestion systems were determined. We aim to reveal the effects of the inocula on the diversity and composition of bacterial and archaeal communities and the mechanism underlying the impact on biogas production in the field. The following results were obtained. (1) Inoculation significantly improved the efficiency of methane production in anaerobic digestion, and the best performance was achieved with treatment A (cleared biogas slurry), which obtained methane production that were 39.59 %, 106.85 %, and 123.00 % higher than in treatment B (pig manure extract), C (cleared biogas slurry + pig manure extract), and the control, respectively. (2) The inocula increased the abundance and diversity of bacterial communities in the early and middle stages and archaeal communities in the early stage. (3) The inocula also had a significant impact on bacterial and archaeal community composition in the early and middle stages. The inocula increased the relative abundance of Aquamicrobium_A, Proteiniphilum, and Cryptobacteroides but reduced the relative abundance of Macellibacteroides, Acinetobacter, and Phocaeicola. The dominant genus of archaeal community was altered from Methanosarcina to Methanocorpusculum. (4) It is concluded that inoculation significantly increase methane production as the inocula improve the abundance and diversity of bacteria and archaea of anaerobic digestion systems in the early and middle stages of digestion, as well as improve the structure and metabolic pathway of archaeal communities.

Applicability of Rice Husk Residue Generated by the Silica Extraction Process to Anaerobic Digestion for Methane Production

Rice husks are a feedstock of biogenic silica because of their high silica content. After silica extraction, a solid residue comprising mostly carbohydrates is present. Solid residue valorization is important for closed-loop systems using rice husk and has minimal negative environmental impacts. In this study, we used solid rice husk that was generated by silica extractionto anaerobic digestion for producing biomethane. The rice husk residue was characterized in terms of total solids, volatile solids, pH, composition, and particle size. Changing the characteristics increased biogas production by 2.48-fold compared to that of raw rice husk. The residue produced 166.4 mL-biogas g−1 vs. and 100.4 mL CH 4 g−1 VS, much more than previously reported. Microbial community analysis, which was conducted to investigate the biological reasons for increased biogas and methane, found increased Bacteroidetes levels in the rice husk samples. Among archaeal communities, Bathyarchaeota was more abundant in all rice husk samples than in the inoculum. The rice husk residue contained more operational taxonomic units than other samples. These changes in the microbial community significantly influenced the anaerobic digestion of the rice husk residue and improved methane production. Our findings provide a basis for the cleaner utilization of rice husk residue to produce renewable energy.

Effects of cold isostatic press pretreatment of rice straw on microstructure and efficiency of anaerobic digestion for methane production

In this study, rice straw was pretreated using by cold isostatic pressure to disrupt its microstructure and improve the performance of anaerobic digestion, and the optimal process parameters were optimized using the response surface methodology. The results showed that cold isostatic pressure pretreatment under optimal conditions (pressure of 400 MPa and holding time of 9 min) was effective in disrupting the structure of rice straw and improving its biodegradability. The cumulative methane production of the cold isostatic pressure pretreatment group increased by 76% compared to the untreated group. In addition, microbial community analysis showed that the relative abundance of Firmicutes, Halobacterota, DMER64 and Methanosaeta was higher in groups pretreated with cold isostatic pressure than in untreated groups. This study demonstrated the potential of pretreatment of rice straw with cold isostatic pressure to increase methane production during anaerobic digestion.

Effects of magnetite on nitrate-dependent anaerobic oxidation of methane in an anaerobic membrane biofilm reactor: Metatranscriptomic analysis and mechanism prediction

Magnetite has been widely reported to accelerate methane production in anaerobic digestion. However, the effects of magnetite on the process of methane consumption, specifically nitrate-dependent anaerobic oxidation of methane (AOM), and its underlying mechanisms remain poorly understood. In this study, the addition of magnetite significantly improved the removal of nitrate in an anaerobic membrane biofilm reactor (AnMBfR) (25.2 ± 2.5 mg NO3−-N/L/d) compared to that in an AnMBfR without magnetite (8.7 ± 4.2 mg NO3−-N/L/d). Metatranscriptomic analysis showed that genes encoding type IV pilus assembly proteins were highly expressed in nitrate/nitrite-reducing bacteria in the biofilms of the two AnMBfRs. Specifically, the transcript abundance of these genes was observed to be higher in the presence of magnetite (gene expression levels, log2FPKM = 13.08) compared to the absence of magnetite (12.04). Biofilms with magnetite exhibited a linear increase in electron transfer resistance with increasing temperature, and the decrease in pH significantly increased their conductivity, indicating a metallic-like conductivity. Contrarily, an increase in temperature or decrease in pH did not influence the resistance or conductivity of biofilms without magnetite. Some of these cytochrome c (CYC)-like proteins were electrically active in the electrochemical Fourier transform infrared spectra and were closely associated with the outer-membrane multi-heme c-type cytochromes (OMCs). The intensities of the OMC-like bands in the surface-enhanced resonance Raman spectra with biofilms in the presence of magnetite were higher than those without magnetite. This study suggests that magnetite promotes the nitrate-dependent AOM process because of the formation of methanotrophic consortia based on direct interspecies electron transfer (DIET).

Granular activated carbon remediates antibiotic resistance propagation and methanogenic inhibition induced by polystyrene nanoplastics in sludge anaerobic digestion

Nano/microplastics (NPs/MPs) in sewage sludge can induce oxidative stress to the anaerobic digestion (AD) and also proliferate antibiotic resistance genes (ARGs). Recently, granular activated carbon (GAC) has been used as an additive to enhance methane production in AD via direct interspecies electron transfer (DIET); however, its impact on AD exposed to NPs/MPs is yet to be studied. This study examined the effect of GAC (5 and 15 g/L) on sludge AD exposed to 150 µg/L of polystyrene nanoplastics (PsNPs). PsNPs decreased methane yield by 32.3% due to elevated levels of reactive oxygen species. However, GAC addition counteracted this adverse effect and improved methane production, attributed to the potential enrichment of DIET-active microbes and the adsorption of PsNPs by GAC. Moreover, GAC reduced the total abundance of ARGs, which was increased by PsNPs exposure. Thus, GAC can provide dual benefits in mitigating methanogenic inhibition caused by PsNPs and ARG spread.

Enhancement of methane production in anaerobic digestion of high salinity organic wastewater: The synergistic effect of nano-magnetite and potassium ions

During high salinity organic wastewater (HSOW) anaerobic digestion treatment, the process of methanogenesis can be severely inhibited in the high salinity environment, and the accumulation of volatile organic acids (VFAs) leads to failure of the anaerobic reaction. In this study, nano-magnetite and KCl were adopted to alleviate the inhibitory effect of high salinity and enhance the HSOW anaerobic digestion performance. The result showed that, under the optimal dosage of 200 mg/L, nano-magnetite addition promoted the anaerobic digestion performance, and the methane production increased by 11.06%. When KCl was added with a dosage of 0.174%, the methane production increased by 98.37%. The simultaneous addition of nano-magnetite (200 mg/L) and KCl showed a synergistic effect on enhancing HSOW anaerobic digestion performance, and the methane production increased by 124.85%. The addition of nano-magnetite and KCl promoted the conversion of VFAs, especially accelerated the degradation of propionic acid and butyric acid, also it promoted the activity of acetate kinase, dehydrogenase and F420, and thereby enhanced the methanogenesis process. This study could provide a new method for enhancing the anaerobic digestion of HSOW.

Enhanced stability of food waste anaerobic digestion under low inoculum to substrate ratio by using biochar

ABSTRACT The influence of biochar on anaerobic digestion (AD) of organic waste have been widely studied. However, the effect of biochar on the mitigation of acidification and subsequently the stimulation of methanogenesis recovery during mono food waste (FW) digestion process under a low inoculum to substrate (I/S) ratio (i.e. a high organic loading) is rarely investigated. In this study, the benefit of biochar with respect to methane production from FW was explored in a mono FW AD system with four different additional amounts of biochar, i.e. 0, 5, 10 and 15 g/L. Results revealed that biochar boosted methane production in AD at a low I/S ratio by 390–530% through stimulating methanogenic activity, improving organics removal and enhancing process stability. The biochar dosage of 10 g/L demonstrated the highest biodegradability of 92.3% and the highest specific methane production of 553.0 mL/g VSremoved among all groups. Without biochar addition, volatile fatty acids (VFAs) accumulated to 20 g/L and the highest total ammonium-N (TAN) was > 1200 mg/L. The suppression of methanogenesis was significantly correlated with VFA and TAN (p < 0.05). Therefore, biochar addition presented a positive effect on VFAs degradation and buffering capacity which could be an effective approach to enhance methane production from FW digestion at a low inoculum to substrate ratio without the fear of system failure.

Exploring the stability of an A-stage-EBPR system for simultaneous biological removal of organic matter and phosphorus

This work evaluates the performance and stability of a continuous anaerobic/aerobic A-stage system with integrated enhanced biological phosphorus removal (A-stage-EBPR) under different operational conditions. Dissolved oxygen (DO) in the aerobic reactor was tested in the 0.2–2 mgDO/L range using real wastewater amended with propionic acid, obtaining almost full simultaneous COD and P removal without nitrification in the range 0.5–1 mgDO/L, but failing at 0.2 mgDO/L. Anaerobic purge was tested to evaluate a possible mainstream P-recovery strategy, generating a P-enriched stream containing 22% of influent P. COD and N mass balances indicated that about 43% of the influent COD could be redirected to the anaerobic digestion for methane production and 66% of influent NH4+-N was discharged in the effluent for the following N-removal B-stage. Finally, when the system was switched to glutamate as sole carbon source, successful EBPR activity and COD removal were maintained for two months, but after this period settleability problems appeared with biomass loss. Microbial community analysis indicated that Propionivibrio, Thiothrix and Lewinella were the most abundant species when propionic acid was the carbon source and Propionivibrio was the most favoured with glutamate. Thiothrix, Hydrogenophaga, Dechloromonas and Desulfobacter appeared as the dominant polyphosphate-accumulating organisms (PAOs) under different operation stages.

Enhanced methane production in anaerobic digestion: A critical review on regulation based on electron transfer

Anaerobic digestion (AD) is a potential bioprocess for waste biomass utilization and energy conservation. Various iron/carbon-based CMs (e.g., magnetite, biochar, granular activated carbon (GAC), graphite and zero valent iron (ZVI)) have been supplemented in anaerobic digestors to improve AD performance. Generally, the supplementation of CMs has shown to improve methane production, shorten lag phase and alleviate environmental stress because they could serve as electron conduits and promote direct interspecies electron transfer (DIET). However, the CMs dosage varied greatly in previous studies and CMs wash out remains a challenge for its application in full-scale plants. Future work is recommended to standardize the CMs dosage and recover/reuse the CMs. Moreover, additional evidence is required to verify the electrotrophs involved in DIET.

Urine pretreatment significantly promotes methane production in anaerobic waste activated sludge digestion

Methane production of waste activated sludge (WAS) in anaerobic digestion is hindered due to the rate-limited hydrolysis process and the low methane potential of WAS. Pretreatment of WAS is a common and appealing strategy to improve methane production in anaerobic digestion. In this study, we proposed to use urine, an easily obtained human waste with high ammonium concentration and pH, as a novel pretreatment strategy for anaerobic WAS digestion. Urine pretreatment at levels of 5–30 % (Vurine/Vurine+WAS) could substantially enhance methane production by 5–35 % in biochemical methane potential (BMP) tests, with the highest methane production of 179.6 ± 3.3 mL/g volatile solids (VS) achieved under the highest level of urine (i.e. 30 % urine addition). Based on the model analysis, the biochemical methane potential (B0) and hydrolysis rate of WAS (k) rose from 131.9 mL/g VS and 0.19 d−1 in the control without pretreatment to 136.3–178.2 mL/g VS and 0.22–0.30 d−1, respectively, after the urine pretreatment (5–30 % addition). Urine pretreatment with 5–30 % addition also improved the degradation extent (Y) of WAS by 3–35 %. The promising results indicate that urine pretreatment in anaerobic digestion is a promising technology to improve the efficiency of anaerobic digestion with environmental and economic benefits.

New insights into wine waste management: Zero waste discharge-driven full energy/resource recovery strategy

This study developed an innovative strategy for wine waste management towards zero-waste discharge with full energy and resource recovery. Waste grape seed, as one of the most typical wine wastes, was first treated by aqueous ethanol, after that the grape seed oil and polyphenols were recycled, and the rest of solid residues were subjected to anaerobic digestion for methane production. It was found that the obtained grape seed oil was rich in about 82% of unsaturated fatty acids, of which all the parameters met the vegetable oil standard, this in turn suggested that the harvested grape seed oil was a high value-added and safe edible oil. While the produced polyphenols with outstanding antioxidant and antibacterial activity respectively verified by DPPH and ABTS radical scavenging assays and MIC and agar well diffusion assays could be used as food additives. Furthermore, the generated digestate after anaerobic digestion could meet all the requirements for organic fertilizer standard. Based on the obtained data from aqueous ethanol extraction and anaerobic digestion, it was estimated that about 122.2 kg of grape seed oil, 23.8 kg of polyphenols, 109.3 m3 of methane and 352.3 kg of organic fertilizer could be produced from 1 ton of grape seed by adopting the proposed strategy. As such, it is expected that this study may lead to the paradigm shift in wine waste management with the ultimate target of zero waste discharge.

Uncovering the effect of polyethyleneimine on methane production in anaerobic digestion of sewage sludge

The potential effect of polyethyleneimine as a flocculant on anaerobic digestion of sludge was investigated. Polyethyleneimine above 12 g/kg total suspended solids inhibited the entire anaerobic digestion process including solubilization, hydrolysis, acidification, and methanogenesis. The addition of 24 g/kg total suspended solids polyethyleneimine reduced methane production from 167 ± 5 L/kg volatile suspended solids in the control reactor (without polyethyleneimine) to 141 ± 5 L/kg volatile suspended solids. Polyethyleneimine bound to extracellular polymeric substances, thus enhancing sludge agglomeration and hindering the release of organics. Meanwhile, the reduction of cytochrome C impeded electron transport, consequently curbed direct interspecies electron transfer. The adsorption of carbon dioxide by amine groups also hampered methane conversion. This study elucidated the concept that polyethyleneimine reduces mass transfer in anaerobic digestion, providing new insights into the potential behavior of flocculants in sludge treatment.

Automated machine learning-based prediction of microplastics induced impacts on methane production in anaerobic digestion

Microplastics as emerging pollutants have been heavily accumulated in the waste activated sludge (WAS) during biological wastewater treatment, which showed significantly diverse impacts on the subsequent anaerobic sludge digestion for methane production. However, a robust modeling approach for predicting and unveiling the complex effects of accumulated microplastics within WAS on methane production is still missing. In this study, four automated machine learning (AutoML) approach was applied to model the effects of microplastics on anaerobic digestion processes, and integrated explainable analysis was explored to reveal the relationships between key variables (e.g., concentration, type, and size of microplastics) and methane production. The results showed that the gradient boosting machine had better prediction performance (mean squared error (MSE) = 17.0) than common neural networks models (MSE = 58.0), demonstrating that the AutoML algorithms succeeded in predicting the methane production and could select the best machine learning model without human intervention. Explainable analysis results indicated that the variable of microplastic types was more important than the variable of microplastic diameter and concentration. The existence of polystyrene was associated with higher methane production, whereas increasing microplastic diameter and concentration both inhibited methane production. This work also provided a novel modeling approach for comprehensively understanding the complex effects of microplastics on methane production, which revealed the dependence relationships between methane production and key variables and may be served as a reference for optimizing operational adjustments in anaerobic digestion processes.